The present invention relates to a new method of chromatography hereafter referred to as Dynamic column Liquid Chromatography (DCLC) for separating two or more components. More particularly the invention relates to a new method for separation of two or more compounds present in a solution using a moving bed chromatographic system.
Chromatography is a term that describes a number of physical methods used in chemistry and biology to separate and identify mixtures of chemical compounds. The principle behind all chromatography variants lies in the repeated subjection of a mixture of chemical compounds to extraction by liquid or adsorption on a solid surface. The mixture is moved physically over a stationary phase (bed or column), which may be either a solid or a liquid immobilized in the pores of a solid (located in said bed or column). The separation of chemical compounds by chromatography may make use of one or more of the following physico-chemical forces, depending upon the particular chromatographic system:
(a) Differences in adsorption to the porous medium, the so called sorbent. PA0 (b) Differences between the relative solubilities of a liquid coating the inert medium (stationary phase) and the liquid, called mobile phase, percolating through the porous column. PA0 (c) Differences in ion exchange with the sorbent. PA0 (d) Differences in molecular size as the solution percolates through a gel of very small size. PA0 (a) In contrast to the gravitational flow which exists in the conventional chromatography, in the dynamic column liquid chromatography some pressure is inherently exerted in the adsorbent bed, which imparts a better resolution in the separation of the constituents. PA0 (b) The method is very rapid, also as a result of the intrinsic pressure exerted in the system. PA0 (c) The method requires less eluent than in the conventional chromatography. PA0 (d) The presence of intrinsic pressure in the dynamic chromatography system enables to utilize an adsorbent with smaller particles size than in the conventional chromatography, which enables a higher sensitivity. PA0 (a) Potential achievement of greater separation efficiences for complex mixtures and hard to resolve solutes. PA0 (b) A substantial decrease in consumption of eluent.
Chromatography is also named preparative chromatography when it is used for isolation of a fraction from a mixture for further uses such as spectroscopy, identification, synthesis for research or commercial purposes.
The original work on chromatography is based on differences in adsorption over an inert material packed in a column. The separation of components, known also as partition chromatography is based on the relative solubilities in the solvent which is passed over the column. The resolution obtained in this chromatography depends upon the pH and ionic strength of the solvent--the mobile phase--and the relative solubilities of the constituents in the two phases; the various materials may be eluted with an appropriate solvent and the liquid fractions collected in a series of tubes and subsequently analysed by chemical or physical methods. Thin layer chromatography (TLC) and paper chromatography, are based on differences between the relative adsorption of a component onto an inert medium. In TLC, the stationary phase consists of a thin layer of a finely divided substance applied to a sheet or plastic backing or to a glass plate. Sorbents commonly used, and commercially available as finished plates, include alumina, silica gel and cellulose. In paper chromatography, the mobile phase may move upwards by capillary action, so called ascending chromatography, or downwards by gravity, so called descending chromatography.
Ion exchange chromatography, involves the separation of molecules based on their ionic charge. The sorbent or stationary phase, consists of polymers with covalently bound ions. In cation exchange resins, the tightly bound ions are negatively charged and are associated with positive ions that are loosely attached by electrostatic charges. The positively charged substances to be separated from a mixture are first adsorbed to the sorbent, displacing the cations present in the resin. The solution is buffered at a pH that will facilitate the binding and then eluted with the same buffer to remove the non-binding fractions of the solution. An anion exchanger operates in exactly the same way, except that its covalently bound ions are oppositively charged to attract the anions from the solution.
The separation based on differences in molecular size is encountered in gel filtration, also known as molecular sieve chromatography. This method separates molecules according to their size, although the shape of the molecule affects the filtration to some extent. The gels are in the form of beads containing a network of openings of pores in which small molecules may be entrapped. The vast commercial interest in chromatography in general and preparative liquid chromatography in particular, is manifested by the large number of publications suggesting various microparticulate column packings and prepacked columns claiming to obtain better separation than the known adsorbents used in this field.